Post-exposure immunoprophylaxis will be achieved only when the following requisite characteristics of an HIV monoclonal antibody are realized: neutralization of globally diverse HIV isolates; lack of toxicity; excellent pharmacokinetics; and reproducible properties as determined by sensitive in vitro assays. The goal of the proposed project is to address these factors by developing several improved approaches to neutralizing antibody assay methodology, and by applying the improved assays to the characterization of a novel monoclonal antibody which inhibits a wide range of HIV variants by targeting the host cell. Other monoclonal antibodies having broad HIV neutralizing activities will also be included for characterization by the improved assays so as to allow formation of optimal monoclonal antibody mixtures, if required. In a long-term collaboration between the Principal Investigator and United Biomedical, Inc., a proprietary anti-host cell monoclonal antibody ("B4") has been developed and partially characterized. B4 is directed against a complex comprising CD4 in association with domains of multiple chemokine receptors. In preliminary studies, it has successfully neutralized SIV, SHIV, HIV-2 and representatives of multiple subtypes (clades) of HIV-1, even when the antibody is added after infection of the cells by a PBMC-grown primary isolate. The antibody, which is not cytotoxic in vitro, has also protected humanized mice when added up to four hours after HIV challenge. In the proposed studies, the murine antibody will be humanized in collaboration with the MRC Collaborative Centre in London, subjected to complete toxicological testing, and, in the final year of the project, evaluated in human clinical trials. The improved neutralizing antibody assays will be used to further characterize both the murine and human forms of the antibody to help monitor the clinical trial.